A chemical reaction is a process in which substances are transformed. One or more reaction products form from one or more starting substances (educts or reactants). The chemical or physical properties of the product are different from those of the educt.
In an enzymatically catalyzed reaction, an enzyme participates in the initiation, acceleration or control of the chemical reaction.
Many diagnostically relevant parameters are enzymes, reactants, reaction products or modulators of enzyme activity. An activity modulator is to be understood as a substance that is able to influence the activity of an enzyme, as inhibitor, activator, agonist, antagonist or cofactor.
An example of a diagnostically relevant enzyme is thrombin, a blood clotting factor in the serine protease group, determination of the activity of which in a patient's plasma sample can provide information about the status of the patient's blood coagulation. Another example is the blood clotting factor VII activating protease (FSAP), determination of the activity of which can for example provide information about a patient's risk of developing a thrombosis.
The activity of an enzyme is usually determined from the reaction of a more or less specific substrate. The known methods are divided into homogeneous methods (without separation steps) and heterogeneous methods (with one or more separation steps).
In heterogeneous methods, the substance to be detected is enriched. That is, the sample being investigated is submitted to a process by which the concentration of other constituents of the sample is reduced relative to the substance to be detected or they are removed completely. Affinity techniques, for example, are used for this.
In contrast, in homogeneous methods the samples are investigated in the assay directly. Separation processes, for example washing steps, are not used.
Examples of homogeneous methods of determining the activity of an enzyme are e.g. thrombin activity assays, such as the ETP assay, as described in EP-A2-0420332. The thrombin in a plasma sample is activated, and a chromogenic peptide substrate is mixed with the sample, and its specific cleavage by thrombin is measured photometrically. This homogeneous assay format is not, however, suitable for determining the activity of enzymes with low substrate specificity, as the lack of substrate specificity means that the substrate can also be converted by other enzymes from the sample. Homogeneous methods are also unsuitable for the determination of enzymes for which only substrates with inadequate analytical sensitivity are available. Furthermore, this homogeneous assay format as a rule is also unsuitable for determining the activity of enzymes with low volume activity.
The volume activity describes the catalytic activity of an enzyme per unit volume. The catalytic activity is defined by the amount of substrate that is reacted in unit time, or the amount of product that is formed in unit time. The volume activity is often stated in μmol (substrate degradation or product formation) per minute and per liter, i.e. μmol/min*L. For μmol/min, usually the term unit (U) or catalytic unit (kU) is used. According to the International System of Units (SI) the unit katal (kat) should be used, i.e. mol/sec.
Along with the volume activity of an enzyme in a sample, in particular the specific activity of an enzyme describes its activity state. The specific activity is found from the ratio of the catalytic activity of an enzyme to the amount of the enzyme present, i.e. μmol/min*g (U/g). The specific activity can for example provide information about the degree of activation, the degree of degradation or the inhibition or activation of an enzyme.
If a sample to be investigated contains other enzymes, which also transform the substrate used, which may be the case in particular in complex samples such as samples of body fluids, specific determination of the desired enzyme activity cannot be carried out directly in the sample without a suitable highly specific substrate.
In the prior art, this problem can be solved for example by determining, instead of the enzyme activity, the enzyme concentration or antigen concentration by means of a specific assay, for example by means of an immunoassay. The antigen concentration is correlated with the expected enzyme activity. Estimating the enzyme activity from the enzyme concentration is unsuitable if the activity state of the enzyme is for example influenced by activity modulators. Therefore the activity of an enzyme or its activity state cannot always be determined with certainty.
Furthermore, in the prior art the problem is also usually solved by using heterogeneous assay systems. In this case the enzyme to be detected is separated from the other constituents of the sample, before the enzyme activity is determined. Separation is usually effected by contacting the sample with a solid phase that has a specific binding affinity for the enzyme to be detected, and subsequent separation of the solid phase.
The disadvantage of these heterogeneous methods is that they include at least one separation step, and perhaps even additional washing steps. These additional steps make automation more difficult and lengthen the processing time. The processing time is also longer because the sample may be incubated with the solid phase for a certain length of time, to enable the enzyme to be detected to bind to the solid phase. Another disadvantage is that the incubation, separation and washing of the solid phase can have an effect on the activity of the enzyme to be detected. For example, conformational changes or degradation of the enzyme can lead to changes in activity. Another disadvantage of the heterogeneous methods is that other constituents of the sample are removed or at least their concentrations are reduced, so that binding partners, cofactors, activators or inhibitors that are intrinsic to the sample, and which influence the activity of the enzyme to be detected, are also removed, so that the enzyme activity or possible binding partners of the enzyme cannot be determined in the physiological context of the sample matrix.